3.3.3　Recovering Functional Antitumor Antibodies

Both tumor-infiltrating and circulating B cells and plasma cells have been shown to be a potent source of antibodies that recognize autologous tumors as well as some heterologous tumors-predominantly those of the same tissue origin.To recover functional antitumor antibodies via high-throughput sequencing, it is crucial to obtain full-length and error-free immunoglobulin sequences and to accurately pair light and heavy chains from each antibody of interest.For the largest intra-tumoral immunoglobulin-producing plasma cell clones, information on full-length IgH and Igκ or Igλ light chains can be extracted from targeted immunoglobulin-sequencing data or RNA-seq data.The latter option was upgraded in MiXCR.The resulting heavy and light chains can be paired on the basis of frequency matching.Because some of the largest IgG1 clonotypes could play an important role in cancer surveillance, the RNA-seq based approach could be directly used in cancer immunotherapy using autologous or allogeneic cytotoxic antibodies or chimeric antigen receptor (CAR) T cells.Implementation of microfluidic emulsion-based approaches can provide a comprehensive analysis of paired heavy and light immunoglobulin chains expressed by hundreds and thousands of individual B cells.The combination of molecular barcoding and droplet barcoding is crucial for providing increased accuracy of analysis.This approach was applied to the analysis of tumor-infiltrating lymphocytes in ovarian cancer, revealing immunoglobulin pairs per 400,000 unsorted tumor cells that entered the emulsion reaction.In general, the rapid development of single-cell analysis approaches, especially those based on emulsion techniques, is revealing new avenues for both immunoglobulin repertoire and transcriptomic profiling of tumor-infiltrating B cells, allowing pairing of heavy and light chains of immunoglobulins as well as linkage of the repertoire with antigenic specificity and the single-cell transcriptome.A remarkable example is a recent work in which New York oesophageal squamous cell carcinoma 1 (NY-ESO-1)-specific antibodies were successfully identified in breast cancer by single-cell analysis of B cells taken from tumor-draining lymph nodes.Several reconstituted antibodies were cloned and shown to be affinity maturated against NY-ESO-1 and were also found in the circulation by affinity chromatography enrichment and mass spectrometry.The microfluidic methods are still relatively laborious and expensive and thus are currently limited to relatively small numbers of samples and cells.Nevertheless, these powerful approaches could help reveal the functional heterogeneity of tumor-infiltrating B cells and suggest rational therapeutic approaches that canalize their functionality towards desirable antitumor action.In the future, direct single cell-based clinical applications may become possible that allow rapid identification of tumor-specific IgG1 antibodies for use in cancer immunotherapy [47, 60-63].(https://www.daowen.com)